Specific sequence amplification of bovine viral diarrhoea virus nucleic acid

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Abstract
Bovine viral diarrhoea virus (BVDV) causes infection of cattle worldwide and is a common contaminant of cell cultures in the laboratory. Methods of diagnosis for BVDV are time-consuming and inconsistent. We describe the development of an in vitro test based on enzymatic DNA amplification withThermus aquaticus DNA polymerase of sequences of BVDV cDNA reverse transcribed from viral RNA. Specific sequences were amplified from infected cell cultures and clinical material using laboratory and field strains of BVDV including both cytopathic and non-cytopathic isolates. Both plus and minus strands of viral RNA can act as suitable templates for cDNA synthesis prior to sequence amplification. Internal restriction digest of amplified sequences and the co-amplification of multiple sequences increased the specificity of the reaction. The significance of the technique in relation to the diagnosis and understanding of strain differences is also discussed.